This invention is related to the characterization of individual chromosomes and, more particularly, to the characterization of individual chromosomes for separation using flow cytometry. This invention was made with government support under Contract No. W-7405-ENG-36 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
The entire set of human chromosomes, 22 autosomes plus the X and Y chromosomes, contains all of the nuclear DNA in human cells, i.e., the human genome. Separating the chromosomes contained in a cell sample provides a convenient way to make chromosome libraries, each of which is a subset of the genome. One convenient separating technique is flow cytometry, or flow sorting, of the chromosomes.
Chromosome sorting by flow cytometry requires that a chromosome or chromosomes to be separated have some identifiable characteristic that can be identified for use in the sorting process. Such chromosome sorting conventionally requires staining a chromosome or chromosomes with two different fluorescent dyes. A typical process is described in J. W. Gray et al., "High-Speed Chromosome Sorting," 23 Science 323-329 (Oct. 1987). A liquid suspension of the chromosomes is stained with two fluorescent dyes, e.g., Hoeschst 33258, which binds preferentially to AT-rich DNA, and chromomycin A.sub.3 (CA3), which binds preferentially to GC-rich DNA. The stained chromosomes pass through two laser beams, one beam to excite each dye to fluorescence. A combination of the fluorescent emission intensities from the two dyes allows many of the chromosomes to be uniquely characterized for separation from other chromosomes, as is fully shown by FIG. 1 herein.
FIG. 1 depicts a conventional bivariate profile for human GM130 chromosomes stained with Hoechst 33258 and CA.sub.3. The combination of fluorescent intensities allows the chromosomes to be karyotyped for separating the chromosomes by flow cytometry, as described by Gray. However, two staining processes must be used for the conventional technique. Further, conventional stains have relatively low quantum efficiencies so that correspondingly high power output lasers are required to obtain sensitive results.
Accordingly, it is an object of the present invention to provide for chromosome karyotyping using only a single fluorescent dye.
Another object of the present invention is to provide sensitive fluorescence signals with relatively low power lasers.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.